Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. Currently, the treatment of diabetes, both type 1 diabetes and type 2 diabetes, relies to an increasing extent on insulin treatment.
Within the last decade a number of human insulin analogues have been designed for particular profiles of action, i.e. fast acting or prolonged action. For instance, insulin Detemir® is a long-acting human insulin analogue for the maintenance of the basal level of insulin. The presence of a fatty acid (myristic acid) covalently bound to the lysine in position B29 delays the action of the insulin.
Despite the recent improvements in insulin analogues available on the market, there is a constant need to enlarge and diversify the existing panel of insulin analogues, exhibiting various pharmacological properties, such as stability and action profiles, while maintaining an efficient insulin activity, in order to fulfil the variety of the specific needs of patients.
Lysine residues have been used as attachment sites for chemical compounds onto insulin molecules. However, the method for preparation of insulin derivatives using lysine as anchoring points presents limitations and is not easy to handle. Indeed, lysine does not allow a site specific attachment onto insulin molecules, as the insulin molecules also comprise two N-terminal α-amino groups that react under similar conditions to ε-amino group of lysine. The reaction with additional chemical groups may result in a heterogenous mixture of one or more of such chemical groups attached at various sites on the insulin. In addition, it is not possible to have an internal lysine in the insulin molecule, when an A. lyticus protease (ALP) which cleaves after lysine residues is used in the processing of insulin. Therefore, the attachment site of a chemical group is restricted to lysine residues in the C terminal end of the A-chain and/or the B-chain.
It has been suggested to use cysteine as anchoring point. Kuo et. al. suggest, in the patent document WO2010/033220 related to therapeutic peptides intended for pulmonary administration, to replace any one of the first four amino acids of the B-chain of insulin with a cysteine residue and then to react such cysteine residue with an activated polyethylene glycol (PEG) that is specific for reaction with thiol groups, e.g., a N-maleimidyl polymer. However, it is known that the addition of cystein residues into insulin generally destabilises the molecule, and Kuo et. al. do not disclose a method to prepare such peptides and do not prove the feasibility and the efficiency of such compounds.
Therefore, there is still a need for new insulin analogues and derivatives, new anchoring sites onto insulin molecules, and improved methods for their preparation.